RESUMEN
Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2â³ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.
Asunto(s)
Aminoglicósidos , Antígenos O , Pseudomonas aeruginosa , Trisacáridos , Pseudomonas aeruginosa/inmunología , Antígenos O/química , Antígenos O/inmunología , Trisacáridos/química , Trisacáridos/inmunología , Trisacáridos/síntesis química , Aminoglicósidos/química , Aminoglicósidos/síntesis química , Aminoglicósidos/inmunologíaRESUMEN
Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.
Asunto(s)
Antígenos O , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antígenos O/química , Antígenos O/inmunología , Antígenos O/análisis , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/clasificación , Escherichia coliRESUMEN
Synthesis of the pentasaccharide repeating unit of the cell O-polysaccharide produced by Salmonella milwaukee O:43 strain (group U) has been achieved in very good yield adopting a convergent stereoselective [3 + 2] block glycosylation strategy. Thioglycosides and glycosyl trichloroacetimidate derivative were used as glycosyl donors in the presence of a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic activator and TMSOTf as trichloroacetimidate activator respectively. The stereochemical outcome of all glycosylation reactions was excellent.
Asunto(s)
Secuencia de Carbohidratos , Pared Celular , Antígenos O , Antígenos O/química , Pared Celular/química , Salmonella/química , Glicosilación , Oligosacáridos/química , Oligosacáridos/síntesis química , Succinimidas/química , Tioglicósidos/química , Tioglicósidos/síntesis química , Estereoisomerismo , Compuestos de Trimetilsililo/química , Acetamidas , Mesilatos , CloroacetatosRESUMEN
Fusobacterium nucleatum, a colorectal-cancer-associated oncomicrobe, can trigger or accelerate numerous pathologies. We report the first synthesis of a conjugation-ready disaccharide containing six amino groups from F. nucleatum ATCC 23726 O-antigen. Rare 2,3-diamido-d-glucuronic acid amide and 2-acetamido-4-amino-d-fucose were synthesized from d-glucosamine through configuration inversion, nucleophilic substitution, C6 oxidation, and C6 deoxygenation. A judicious choice of protecting groups and reaction conditions enabled the selective installation of N-acetyl, N-propanoyl, N-formyl, and carboxamido groups.
Asunto(s)
Fusobacterium nucleatum , Antígenos O , Fusobacterium nucleatum/química , Antígenos O/química , Estructura Molecular , Disacáridos/química , Disacáridos/síntesis químicaRESUMEN
Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.
Asunto(s)
Glicosiltransferasas , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosilación , Citrobacter/metabolismo , Citrobacter/genética , Antígenos O/metabolismo , Antígenos O/química , Polisacáridos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Polisacáridos Bacterianos/metabolismoRESUMEN
Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: â2)-ß-d-Fucf-(1â3)-ß-d-Fucp-(1â. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: â2)-ß-d-Glcp-(1â. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.
Asunto(s)
Lipopolisacáridos , Ochrobactrum , Lipopolisacáridos/química , Fucosa/química , Antígenos O/química , BacteriasRESUMEN
Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. â2)-α-l-Rhap-(1â2)-α-l-Rhap-(1â3)-α-l-Rhap-(1â3)-ß-d-Galp-(1â.
Asunto(s)
Halomonas , Lipopolisacáridos , Polisacáridos/química , Espectroscopía de Resonancia Magnética , Antígenos O/químicaRESUMEN
Klebsiella pneumoniae is one of the priority objects for the development of new therapies against infections. The species has been perceived as of limited variety of O antigens (11 O serotypes identified to date). That trait makes lipopolysaccharide an attractive target for protective antibodies. Nowadays, K. pneumoniae O antigens encoding genes are often analysed by bioinformatic tools, such as Kaptive, indicating higher actual diversity of the O antigen loci. One of the novel K. pneumoniae O loci for which the antigen structure has not been elucidated so far is OL101. In this study, four clinical isolates predicted as OL101 were characterized and found to have the O antigen structure composed of ß-Kdop-[â3)-α-l-Rhap-(1â4)-α-d-Glcp-(1â]n, representing a novel serotype O13. Identification of the ß-Kdop terminus was based on the analysis of the complete LPS molecule by the HR-MAS NMR spectroscopy. The bioinformatic analysis of 71,377 K. pneumoniae genomes from public databases (July 2023) revealed a notable OL101 prevalence of 6.55 %.
Asunto(s)
Infecciones por Klebsiella , Antígenos O , Humanos , Antígenos O/genética , Antígenos O/química , Klebsiella pneumoniae/genética , Serogrupo , Lipopolisacáridos/químicaRESUMEN
The aim of the study was the serological and structural characterization of the lipopolysaccharide (LPS) O antigen from P. mirabilis Dm55 coming from the urine of a patient from Lodz. The Dm55 LPS was recognized in ELISA only by the O54 antiserum, suggesting a serological distinction of the Dm55 O antigen from all the 84 Proteus LPS serotypes described. The obtained polyclonal rabbit serum against P. mirabilis Dm55 reacted in ELISA and Western blotting with a few LPSs (including O54), but the reactions were weaker than those observed in the homologous system. The LPS of P. mirabilis Dm55 was subjected to mild acid hydrolysis, and the obtained high-molecular-mass O polysaccharide was chemically studied using sugar and methylation analyses, mass spectrometry, and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The Dm55 O unit is a branched three-saccharide, and its linear fragment contains α-GalpNAc and ß-Galp, whereas α-GlcpNAc occupies a terminal position. The Dm55 OPS shares a disaccharide epitope with the Proteus O54 antigen. Due to the structural differences of the studied O antigen from the other described Proteus O polysaccharides, we propose to classify the P. mirabilis Dm55 strain to a new Proteus O85 serogroup.
Asunto(s)
Lipopolisacáridos , Proteus mirabilis , Animales , Humanos , Conejos , Lipopolisacáridos/química , Serogrupo , Antígenos O/química , Secuencia de Carbohidratos , CarbohidratosRESUMEN
Pectobacterium brasiliense is a widely distributed phytopathogenic bacterium that causes diseases such as soft rot and blackleg, leading to significant yield losses in potatoes as well as other vegetables and ornamental plants. Lipopolysaccharide (LPS) is an important virulence factor that plays an essential role in colonisation of plant tissues and overcoming the host defence mechanisms. The O-polysaccharide from the LPS of P. brasiliense strain NCPPB 4609TS (=CFBP 6617TS = LMG 21371TS = IFB5390) was structurally characterised using spectroscopic techniques and chemical methods. The analyses revealed that the polysaccharide repeating unit consists of Gal, GlcN and an unusual 3-amino-3,6-dideoxyglucose decorated with (R)-3-hydroxybutyric acid according to the structure shown below: In addition, another polysaccharide was isolated from bacterial cells, analysis of which led to the identification of an enterobacterial common antigen, containing N-acetyl-d-glucosamine, N-acetyl-d-mannosaminouronic acid, and 4-acetamido-4,6-dideoxy-d-galactose.
Asunto(s)
Antígenos O , Pectobacterium , Antígenos O/química , Lipopolisacáridos/químicaRESUMEN
Herein, we report the total synthesis of the trisaccharide repeating unit of the O-antigen of Actinobacillus actinomycetemcomitans serotype f. The trisaccharide comprising of α-(1-2) and α-(1-3)-linked L-rhamnopyranosides backbone with the latter rhamnose containing a branching N-acetyl-d-galactosaminopyranoside at the C2-O via a ß-glycosidic bond was synthesized by two methods. Initially, the protected trisaccharide has been synthesized by step-wise assembly of the monosaccharide building blocks and subsequently the former was synthesized by the one-pot assembly of the latter components. The synthesized trisaccharide contains an aminoethyl linker appended as an O-glycoside at the reducing end, thereby providing scope for further conjugation for different applications.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Antígenos O , Antígenos O/química , Aggregatibacter actinomycetemcomitans/química , Serogrupo , Monosacáridos , Glicósidos , TrisacáridosRESUMEN
The O-antigen is one of the outermost surface components of Gram-negative bacteria. Its large structural variation provides the molecular basis for bacterial serological diversity. Here, we established the structure of the O-antigen from an Escherichia coli strain, SD2019180, which appeared to be completely different from the known E. coli serogroups. The O-antigen tetrasaccharide biological repeating unit was identified as â 2)-[ß-d-GlcpA-(1 â 4)]-[α-d-Galp-(1 â 3)]-α-l-Fucp-(1 â 3)-α-d-GlcpNAc-(1 â. Furthermore, we analyzed the O-antigen gene cluster of SD2019180 and confirmed its role in O-antigen synthesis by using deletion and complementation experiments. Our findings indicate that SD2019180 is a novel serogroup of Escherichia coli.
Asunto(s)
Escherichia coli , Antígenos O , Escherichia coli/genética , Escherichia coli/química , Antígenos O/genética , Antígenos O/química , Serogrupo , Familia de MultigenesRESUMEN
Methylphosphorylated mono-, di- and trimannosides structurally related to the lipopolysaccharide (LPS) O-antigens of Klebsiella pneumoniae of serotype O3 were synthesized and conjugated with a biotin tag. The stereo- and regioselective assembly of target carbohydrate chains was conducted using uniform monosaccharide synthetic blocks. After that, a methylphosphate group was introduced by coupling with a methyl-H-phosphonate reagent followed by oxidation and deprotection to give the target oligosaccharides. The 1H and 13C NMR spectra of the obtained compounds showed a good fit with the spectrum of the corresponding natural polysaccharide. The newly prepared biotinylated oligosaccharides along with the previously reported biotinylated glycoconjugates related to galactan I and galactan II of K. pneumoniae LPS were used for the ELISA detection of antibodies in anti-K. pneumoniae rabbit sera. Anti-O3 serum antibodies specifically recognized the synthesized oligosaccharide ligands with terminal methylphosphomannosyl residues, whereas anti-O1 serum antibodies recognized the oligosaccharide related to K. pneumoniae galactan II. The analysis of human sera from patients with confirmed Klebsiella infection also revealed the presence of antibodies against the synthesized oligosaccharides in clinical cases. Thus, the described compounds together with other Klebsiella related antigenic oligosaccharides could be potentially used as molecular probes for K. pneumoniae serological diagnostics development and strain serotyping.
Asunto(s)
Lipopolisacáridos , Antígenos O , Animales , Humanos , Conejos , Antígenos O/química , Klebsiella pneumoniae , Serogrupo , Oligosacáridos , Galactanos , AnticuerposRESUMEN
The aim of this work was to examine the structure and gene cluster of O-OPS of S. xiamenensis strain DCB-2-1 and survey its conceivability for chelating uranyl, chromate and vanadate ions from solution. O-polysaccharide (OPS, O-antigen) was isolated from the lipopolysaccharide of Shewanella xiamenensis DCB-2-1 and studied by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and sugar analysis. The following structure of the brunched pentasaccharide was established: where d-ß-GlcpA(d-Ala) is d-glucuronic acid acylated with NH group of d-Ala. The OPS structure established is unique among known bacterial polysaccharide structures. Interestingly, that dN-(d-glucuronoyl)-d-alanine derivative is not found in bacterial polysaccharides early. The O-antigen gene cluster of Shewanella xiamenensis strain DCB-2-1 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the OPS structure. Based on the analysis of the IR spectra of the isolated polysaccharide DCB-2-1 and the products of its interaction with UO2(NO3)2 ∗ 6H2O, NH4VO3 and K2Cr2O7, a method of binding them can be proposed. Laboratory experiments show that the use of polysaccharide can be effective in removing uranyl, chromate and vanadate from solution.
Asunto(s)
Escherichia coli , Antígenos O , Secuencia de Carbohidratos , Antígenos O/genética , Antígenos O/química , Escherichia coli/genética , Amidas , Cromatos , Vanadatos , Familia de Multigenes , Ácido GlucurónicoRESUMEN
The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined: [Formula: see text] The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes. For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.
Asunto(s)
Aeromonas , Carpas , Oncorhynchus mykiss , Animales , Antígenos O/química , Aeromonas hydrophila/genética , Aeromonas hydrophila/química , Serogrupo , Polonia , Aeromonas/genética , Aeromonas/química , AcuiculturaRESUMEN
Herein, we described the first synthesis of the pentasaccharide and decasaccharide of the A.â baumannii ATCC 17961 O-antigen for developing a synthetic carbohydrate-based vaccine against A.â baumannii infection. The efficient synthesis of the rare sugar 2,3-diacetamido-glucuronate was achieved using our recently introduced organocatalytic glycosylation method. We found, for the first time, that long-range levulinoyl group participation via a hydrogen bond can result in a significantly improved ß-selectivity in glycosylations. This solves the stereoselectivity problem of highly branched galactose acceptors. The proposed mechanism was supported by control experiments and DFT computations. Benefiting from the long-range levulinoyl group participation strategy, the pentasaccharide donor and acceptor were obtained via an efficient [2+1+2] one-pot glycosylation method and were used for the target decasaccharide synthesis.
Asunto(s)
Carbohidratos , Antígenos O , Antígenos O/química , Carbohidratos/química , Oligosacáridos/química , Glicosilación , GalactosaRESUMEN
A straightforward synthesis of the hexasaccharide repeating unit of the O-specific polysaccharide of Salmonella arizonae O62 was achieved in very good yield applying sequential glycosylation strategy. Successful regioselective glycosylation of the di-hydroxylated L-rhamnose moiety allowed achieving the desired compound in minimum number of synthetic steps. TEMPO catalyzed and [bis(acetoxy)iodo]benzene (BAIB) mediated late stage regioselective oxidation of a primary hydroxyl group into carboxylic acid was achieved in the hexasaccharide derivative. The glycosylation steps were high yielding with high stereochemical outcome. The desired hexasaccharide was obtained in 7% over all yield in fourteen steps starting from suitably functionalized monosaccharide intermediates.
Asunto(s)
Antígenos O , Salmonella arizonae , Antígenos O/química , Glicosilación , Monosacáridos , Ramnosa , Oligosacáridos/químicaRESUMEN
Ochrobactrum endophyticum (syn. Brucella endophytica) is an aerobic species of Alphaproteobacteria isolated from healthy roots of Glycyrrhiza uralensis. Here we report the structure of the O-specific polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of the type strain KCTC 42485:â3)-α-l-FucpNAc-(1â3)-ß-d-QuipNAc-(1â2)-ß-d-Fucp3NAcyl-(1â where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The structure was elucidated using chemical analyses along with 1H and 13C NMR spectroscopy (including 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC, HSQC-TOCSY and HSQC-NOESY experiments). To our knowledge the OPS structure is novel and has not been previously published.
Asunto(s)
Antígenos O , Ochrobactrum , Antígenos O/química , Galactosa , Lipopolisacáridos/químicaRESUMEN
Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established. â2)[α-D-Fucp3NRHb-(1â3)]-α-L-Rhap-(1â3)-ß-L-Rhap-(1â4)-α-L-Rhap-(1â3)-ß-D-FucpNAc-(1â The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.
Asunto(s)
Aeromonas , Carpas , Animales , Antígenos O/química , Secuencia de Carbohidratos , Serogrupo , Aeromonas/genética , Aeromonas/química , Familia de MultigenesRESUMEN
Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O antigens are unique among the Proteus O serotypes, which had been described earlier, as they were not recognized in an enzyme-linked immunosorbent assay (ELISA) by a set of Proteus O1-O83 antisera. Additionally, the Kr1 antiserum did not react with O1-O83 lipopolysaccharides (LPSs). The O-specific polysaccharide (OPS, O antigen) of P. mirabilis Kr1 was obtained via the mild acid degradation of the LPSs, and its structure was established via a chemical analysis and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy applied to both initial and O-deacetylated polysaccharides, where most ß-2-acetamido-2-deoxyglucose (N-acetylglucosamine) (GlcNAc) residues are non-stoichiometrically O-acetylated at positions 3, 4, and 6 or 3 and 6, and a minority of α-GlcNAc residues are 6-O-acetylated. Based on the serological features and chemical data, P. mirabilis Kr1 and Ks20 were proposed as candidates to a new successive O-serogroup in the genus Proteus, O84, which is another example of new Proteus O serotypes identified lately among serologically differentiated Proteus bacilli infecting patients in central Poland.